Heart disease is the leading cause of morbidity for both men and women, accounting for nearly 1 in 4 deaths. Consequently, there is an increasing need for strategies to generate human cardiomyocytes that are suitable for studies to benefit our understanding and care for patients with heart disease, including cardiotoxicity testing, drug target identification, and the development of personalized treatment strategies. Using modern differentiation protocols, human pluripotent stem cells (hPSC) are a renewable source for the rapid and large-scale production of cardiomyocytes (hPSC-CM). However, outstanding challenges in the generation of hSPC-CM populations optimal for research and clinical applications include the difficulty in producing pure cultures of cardiomyocytes of a specific subtype and maturation stage. To overcome these limitations, a clinically relevant system that does not require genetic modifications to identify and isolate cardiomyocytes with defined functional properties is needed. Informative cell surface proteins and affinity reagents (e.g. monoclonal antibodies) that permit selection of in vitro derived cells at specific stages of differentiation with known functional properties represent a clinically viable solution to these problems. Analogous to what has been accomplished in the hematopoietic stem cell system, we hypothesize that cell surface proteins can be used to identify and select specific cell types within the context of cardiomyocyte differentiation. In support of our goals, we have used an innovative mass spectrometry approach that enables capture and identification of extracellular domains of cell surface proteins to identify >1300 surface proteins on hPSC-CM. Among these is a novel protein, novel cell surface protein (NCSP), that is robustly expressed in human heart. Phylogenetic analyses indicate NCSP emerged in species with four functional heart chambers and, despite having no predicted structural or functional domains, it?s sequence is highly conserved. We have generated novel anti-NCSP antibodies against unique epitopes present on the extracellular domain of NCSP and applied them to specifically detect it on cardiomyocytes both in vitro and in vivo. Building on these preliminary data, we hypothesize that NCSP is an authentic cardiomyocyte cell surface marker that can be used to identify and sort live cells and has a significant cardiomyocyte function. There are two specific Aims to test this hypothesis: (Aim 1) To test the hypothesis that NCSP is a hPSC- CM cell surface marker which can be used to specifically identify and sort live cardiomyocytes. (Aim 2) To test the hypothesis that NCSP is required for the proper function of hPSC derived cardiomyocytes. Overall, the proposed studies will focus on understanding this in the context of cardiomycyte biology and will define the utility of novel monoclonal antibodies for immunophenotyping. More specifically, we will assess the utility of the antibodies for specifically identifying cardiomyocytes in vitro, will determine if this novel protein is required for in vitro differentiation.